Analysis of Incision Sites Produced by Human Cell Extracts and Purified Proteins during Nucleotide Excision Repair of a 1,3-Intrastrand d(GpTpG)-Cisplatin Adduct

Abstract

Nucleotide excision repair by mammalian enzymes removes DNA damage as part of approximately 30-mer oligonucleotides by incising phosphodiester bonds on either side of a lesion. We analyzed this dual incision reaction at a single 1,3-intrastrand d(GpTpG)-cisplatin cross-link in a closed circular duplex DNA substrate. Incisions were formed in the DNA with human cell extracts in which DNA repair synthesis was inhibited. The nicks were mapped by restriction fragment end labeling and primer extension analysis. Principal sites of cleavage were identified at the 9th phosphodiester bond 3' to the lesion and at the 16th phosphodiester bond 5' to the lesion. The predominant product was found to be a 26-mer platinated oligonucleotide by hybridization to a 32P-labeled complementary DNA probe. Oligonucleotides were formed at the same rate as the 3' cleavage, suggesting that both incisions are made in a near-synchronous manner. There was, however, a low frequency of 5' incisions in the absence of 3' cleavage. The dual incision reaction was reconstituted using the purified mammalian proteins XPA, RPA, XPC, TFIIH, XPG, and a fraction containing ERCC1-XPF and IF7. All of these components were required in order to observe any cleavage.